CA2243509A1 - Anti-idiotypic antibody vaccines - Google Patents
Anti-idiotypic antibody vaccines Download PDFInfo
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- CA2243509A1 CA2243509A1 CA002243509A CA2243509A CA2243509A1 CA 2243509 A1 CA2243509 A1 CA 2243509A1 CA 002243509 A CA002243509 A CA 002243509A CA 2243509 A CA2243509 A CA 2243509A CA 2243509 A1 CA2243509 A1 CA 2243509A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
- C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
- C07K16/4266—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig against anti-tumor receptor Ig
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
This invention provides a vaccine comprising an effective amount of antiidiotypic monoclonal antibody, designated BEC2, an effective amount of an appropriate adjuvant and a pharmaceutically acceptable vehicle and different uses of such vaccine.
Description
CA 02243~09 1998-07-1~
ANTI - IDIOTYPIC ANTTBOD~ VACCINES
This application is a continuation-in-part of United States Patent Application Serial No. 08/619,217, ~iled March 21, 1996, the contents of which are hereby incorporated by reference.
The invention disclosed herein was made with the support o~
the NCI Grant No. PO1-CA33049. Accordingly, the U.S.
Government has certain rights in this invention.
Throughout this application, various publications are referenced by author and date. Full citations for these publications may be ~ound listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Backqround of the }nvention GD3 ganglioside is an appealing target for immunotherapy of melanoma for several reasons. Expression of the GD3 ganglioside on normal tissue is selective and occurs at low concentrations. Expression of the GD3 ganglioside is abundant on most melanoma (Hamilton et al., 1993) and antibodies to GD3 can both block melanoma cell attachment to surfaces (Cheresh, et al., 1986; Chapman, et al., l990), an early critical step in invasion and metastasis, and can inhibit melanoma cell growth in vitro (Dippold, et al., 1984). In animal models, monoclonal antibodies (MAb) R24 against GD3 can inhibit outgrowth of melanoma tumors and treatment of metastasic melanoma patients with the anti-GD3 MAb R24 can result in ma3or tumor shrinkage (Vadhan-Raj, et al., 1988; Caufield, et al., 1990; Raymond, et al., l991;
Creekmore, et al., 1992). However, GD3 is poorly immunogenic, and attempts to immunize patients against GD3 using GD3~ cells, puri~ie~ GD3, or GD3 conjugates have been CA 02243~09 1998-07-1~
unsucces6~ul (Tai, et al., 1985; Livingston, et al., 1989).
United States Patent Number 5,529,922, issued June 25, 1996, the content o~ which is incorporated into this application, discloses an anti-idiotypic monoclonal antibody, BEC2 made against the anti-GD3 monoclonal antibody R24 and a method to produce the same. The anti-idiotypic monoclonal antibody, BEC2 carries the internal image of GD3 and then mimics GD3.
Such monoclonal antibody is a valuable immunological reagent. Patients were immunized with BEC2 administered subcutaneously without an immune adjuvant; only 55% o~
patients developed anti-BEC2 antibodies and only 1/20 developed detectable anti-GD3 antibodies.
Potent immunological adjuvants, as QS21 and BCG, have been used to enhance the immune response to ganglioside and to anti-idiotypic MAb-(Mittelman, et al., 1992). QS21 is a saponin extracted from the bark of the South American soap bark tree Quillaja saponaria Molina with significant adjuvant activity ~Kensil, et al., 1991; Wu, et al., 1992;
Newman, et al., 1992; White, et al., 1991). Because of its wide spread use, high adjuvant effect and low toxicity QS21 is best selected. BCG is an attenuated strain o~
Mycobacterium bovis and is a potent immunological adjuvant that has been used to enhance the immune response to gangliosides (Livingston, et al., 1994) and to anti-idiotypic MAb (Mittelman, et al., 1992).
CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 Summary o~ the Invention The present invention provides a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle that enhance the production of antibodies against GD3.
The present invention also provides a method for ~nh~ncing and stimulating production of antibodies against GD3 in a melanoma subject which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle.
The present invention further provides a method for treating melanomas patients which compri~es administering to the subJect a vacclne comprislng an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle.
The present invention further provides a method for preventing recurrence of melanomas which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle.
In one embodiment of the invention, the invention provides a method for increasing the survival rate of small cell lung ~ cancer patients which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle.
CA 02243~09 l998-07-l~
W097/34635 PCT~Ss7/04590 Brief Description o~ the Fiqures Fiqure 1:
A. Reactivity of immune serum from patie~t 13 immunized with BEC2/BCG against BEC2 (-) and MPC11 (-). After 5 immunizations immune serum is diluted as show~ and tested for binding to BEC2 and MPC11 as described in the Experimental section.
B. Mean reactivity of immune sera against BEC2 (solid bars) or MPCll (open bars) in patients immunized with BEC2/BCG (N=14) or BEC2/QS21 (N=6). Each serum sample is tested at a dilution that resulted in absorbance readings of approximatel~ 0.5 against BEC2-coated plates. Error bars represent standard deviation of the mean.
C. Binding of pre-absorbed serum ~rom patient 13 immunized with BEC2/BCG against BEC2 (-) and MPC11 (-). The same serum as shown in Figure lA (diluted to l:lOO,OO0) is exhaustively absorbed against MPC11-coated agarose beads prior to performing the assay.
Fioure 2:
A. Anti-GD3 antibody titers induced by BEC2/BCG in patient 1. Serological assays were performed as described in the Experimental section. Arrows indicate immunization with BEC2/BCG. In this patient, anti-GD3 reactivity was detected only with alkaline phosphatase-conjugated second antibody against human IgG.
B. Anti-GD3 antibody titers induced by BEC2/BCG in CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 patient 3. Serological assays were per~ormed as described in the Experimental section. Arrows indicate immunization with BEC2/BCG. In this patient, anti-GD3 reactivity was detected only with alkaline phosphatase-conjugated second antibody against human IgG.
C. Anti-GD3 antibody titers induced by BEC2/BCG in patient 13. Serological assays were performed as described in the Experimental section. Arrows indicate immunization with BEC2/BCG. In this patient, anti-GD3 reactivity was detected only with alkaline phosphatase-conjugated second antibody against human IgG.
Figure 3: Overall survival o~ patients immunized with BEC2/BCG (Kaplan-Meier Curve).
Figure 4: Overall survival of SCLC patients immunized with BEC2/BCG ~Kaplan-Meier Curve).
CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 Detailed DescriPtion of the Invention The present invention provides a vaccine comprising an anti-idiotypic monoclonal antibody designated BEC2, an effective amount of an appropriate adjuvant and a pharmaceutically acceptable vehicle. This vaccine is useful for stimulating or enhancing in a subject to which the vaccine is administered, production of antibodies against GD3 ganglioside.
As u_ed herein, the term "anti-idiotypic monoclonal antibody designated BEC2-1 includes the mouse BEC2 antibody, chimeric antibody of the mouse BEC2 antibody, single chain antibody of BEC2 and functional fragments of these antibodies.
Preferably, the chimeric antibody is a humanized BEC2 antibody wherein the framework of the immunoglobin molecule is hllm~n and only the Complementarity Determining Region is from mouse. In addition, the antibody may be a single chain antibody. Functional fragments may be the Fab or Fab' fragments. The method to make the functional fragments, chimeric antibodies or single chain antibody of BE~2 are well-known in the art.
In an embodiment, the effective amount of BEC2 ranges from about O. 5 mg to 25 mg. In another embodiment, the effective amount of BEC 2 is 2.5 mg.
In a separate embodiment, the appropriate adjuvant is BCG.
In a further embodiment, BCG is administered in attenuated doses.
In a preferred em~odiment, the vaccine is administered in at least one site on definite time intervals. In a further embodiment, the vaccine is administered in one 03~ more sites. The administration may be effected intradermally, su~cutaneously, parenterally, or intravenously.
CA 02243jO9 l998-07-lj W097/34635 PCT~S97/04590 The present invention also provides a method for enhancing and stimulating production of antibodies against GD3 in a melanoma subject which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of an appropriate adjuvant and a pharmaceutically acceptable vehicle.
The present invention further provides a method for treating melanoma in a subject which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of an appropriate adjuvant and an pharmaceutically acceptable vehicle.
The present invention further provides a method for preventing recurrence of cancer which comprises administering to a melanoma subject a vaccine comprising an e~fective amount of the anti-idiotypic monoclonal antibody designated BEC2, an e~fective amount of an appropriate adjuvant and an acceptable pharmaceutical vehicle.
In one embodiment of the invention, the invention provides a method for increasing the survival rate of small cell lung cancer patients which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and an pharmaceutically acceptable vehicle.
In another em~odiment of the invention, the invention provides a method for increasing the survival rate o~ small cell lung cancer patients which comprises administering to the subject a vaccine comprising an effectlve amount of the anti-idiotypic monoclonal antibody designated BEC2, an e~fective amount of the appropriate adjuvant and an pharmaceutically acceptable vehicle in combination with a CA 02243~09 l998-07-l~
W097/3463S PCT~97/04590 treatment with a chemotherapeutic agent.
In another embodiment of the invention, the appropriate adjuvant is BCG.
In another embodiment of the invention, the ef~ective amount of the anti-idiotypic monoclonal antibody, BEC2, ranges from about 0.5 mg to about 25 mg.
In another embodiment of the invention, the effective amount of the anti-idiotypic monoclonal antibody, BEC2, is about 2.5 mg.
Different effective amounts of the anti-idiotypic monoclonal antibody, BEC2 thereof may be used according to this invention. A person ordinary skilled in the art can perform simple titration experiments to determine what effective amount is required for effective immunization. An example of such titration experiment is to inject different amounts of the anti-idiotypic monoclonal antibody, BEC2, from 0.5 mg to 32 mg, to make a seroconversion and then ~m; ne the immune response.
In another embodiment of the invention, the adjuvant is BCG
and an effective amount of BCG is about 107 Colony Forming Units (CFU).
Different effective amounts of the appropriate adjuvant thereof may be used according to this invention. A person ordinary skilled in the art can perform simple titration experiments to determine what effective amount is required for effective immunization. An example of such titration experiment is to inject different amounts of the appropriate adjuvant and then examine the immune response.
In another embodiment of the invention, the vaccine comprising 2.5mg of BEC2 and attenuated doses of BCG is CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 injected in weeks 0,2,4,6 and 10.
In another embodiment of the invention, subsequent doses of BCG are 3-fold attenuated.
In another embodiment of the invention, the vaccine is injected intradermally, subcutaneously or intravenously.
Pharmaceutically acceptable carriers are well known to those skilled in the art. Such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the speci~ic experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follow thereafter.
CA 02243~09 l998-07-l~
W097/3463S PCT~S97/0459 Ex~erimental Detail~:
First serie~ of ex~eriment~:
Material and Methods Patients selection Se~uential clinical trials in which melanoma patients free of disease after complete surgical resection but at high risk for recurrence are immunized with BEC2 mixed with BCG
(BEC2/BCG) using 2.5 mg of BEC2. Patients free of disease afte_ resection of AJCC stage II are also eligible if the primary tumor was 24mm deep. Other eligibility re~uirements are: normal WBC and differential count, no previous chemotherapy, immunotherapy, or radiation therapy within the previous month, and no history of other malignancies within the past 5 years. Patients are excluded if they have significant underlying medical problems (e.g. NYHA class III
or IV cardiac disease, active infections requiring antibiotics, or active bleeding), required antihistamines, steroids, or non-steroidal anti-inflammatory drugs, have an immunodeficiency or are asplenic, have a history of metastases to the CNS, or are pregnant or lactating.
Vaccine components BEC2 is purified from virus-free lipoprotein-free cell culture supernatants by Celltech, Ltd (Slough, Bngland) using Protein A affinity chromatography and anion-exchange chromatography. The final product is >95~ pure as determined by SDS-polyacrylamide gel electrophoresis. BEC2 is supplied in sterile phosphate buffered saline at concentration of 2.5 mg/ml in single-dose vials.
BCG (TheraCys~) is purchased from Connaught Laboratories (Swiftwater, PA) in vials containing 3.4 x 108 colony-forming units (CFU)/vial in a ~reeze-dried form. Immediately before administration, BCG is reconstituted with 3.4 ml of sterile diluent and then further diluted in normal saline if CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 necessary. Vaccine is prepared by mixing 1 ml o~ BEC2 (2.5 mg) with O.l ml of the appropriate dilution of BCG
immediately prior to administration.
QS21, supplied by Cambridge Bioscience, Inc. (Worcester, MA), is extracted ~rom Quillaja saponaria Molina tree bark using silica and reverse-phase chromatography as previously described (Kensil, et al., 1991). It is supplied in 2.1 mg vials as a lyophilized powder and once reconstituted with sterile phosphate buffered saline, can be stored at 4~C for not more than 14 days. Vaccine containing QS21 is prepared immediately prior to ~min;stration by mixing 1 ml of BEC2 (2.5 mg) with lO0 ~g o~ QS21. The dose o~ QS21 is selected based on a previous phase I clinical trial (Livingston, et al., 1994).
The vaccine is injected intradermally into multiple rotated sites on weeks O, 2, 4, 6, and lO; injections are given only into limbs in which the regional lymph node basin is intact.
In patients who developed anti-GD3 antibodies, a sixth immunization is offered at a time when the anti-GD3 antibody titers returned to baseline.
The initial immunization contained 2.5 mg BEC2 mixed with 1 x 107 CFU o~ BCG. In the setting of grade III local toxicity, subsequent doses o~ BCG are attenuated 3-~old (i.e. 3 x 106 CFU, 1 x 106 CFU, 3 x 105 CFU, 1 x lO~ CFU, 3 x 104 CFU, 1 x 104 CFU). Patients with a positive PPD (or a hlctory of a positive PPD), are started at an atte~uated BCG
dose of 3 x 106 CFU.
Vaccine is administered as a single subcutaneous injection using the identical schedule and dose o~ BEC2 as ~or the BEC2/BCG vaccine (wee~s O, 2, 4, 6, and lO). Each vaccine consisted o~ 2.5 mg BEC2 and 100 ~g of QS21; the QS21 dose CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 is not attenuated.
Seroloqical evaluat.ion Serum is collected pre-treatment, at the time o~ each immunization, and two weeks following each immunization and stored at -20~C. Serum is tested for the presence of B~C2 antibodies and anti-GD3 antibodies using an ELISA assay as previously described (Chapman, et al., 1994). Internal standards are run with each assay to ensure adequate and reproducible sensitivity. The specificity of the anti-BEC2 response is characterized by comparing the anti-BEC2 IgG
response to the IgG response against MPC11, an isotype-matched mouse MAb (IgG2b) with framework sequences (Ng, et al., 1993~. Ab3 antibodies are detected by their ability to bind BEC2 but not MPC11. The degree of binding of BEC2 versus MPC11 is compared by ELISA using a serum dilution that results in sub-maximal binding to BEC2 (absorbance values o~ approximately 0.5 are used). This is done ~irstly to ensure that the assay is carried out at a serum dilution corresponding to the linear portion of the titration curve for each serum sample and secondly, to st~n~dize the assay allowing the data to be analyzed according to treatment group. The presence of Ab3 antibodies is con~irmed in selected patients by exhaustively absorbing immune serum against agarose beads ~Affigel lO, Bio-Rad Laboratories, ~ercules, CA) coated with MPC11 until reactivity against MPC11 was not detected. The absorbed serum is then assayed ~or binding to BEC2.
A patient is considered to have developed anti-GD3 antibodies if at least two post-treatment sera samples reproducibly demonstrate a 4-fold or greater increase in anti-GD3 reactivity compared to the pre-treatment sample.
This strict criterion was established based on our serological studies in patients immunized with BCG alone to avoid confusion with transient, low-level, non-specific reactivity that can be induced by BCG and other adjuvants.
CA 02243509 l998-07-l5 W097/3463~ PCT~S97/04590 Attempts to characterize further the specificity of anti-GD3 responses employed mixed hemadsorption assays against GD3 allogeneic melanoma cell lines (Vadhan-Raj, et al., 1988) and immuno-thin layer chromatography ~Chapman, et al., 1994).
Ex~erimental Results:
Patient characteristics Fourteen patients are immunized with BEC2/BCG while 6 patients are immunized with BEC2/QS21. Patient characteristics are shown in Table 1. While the two groups of patients are relatively similar, the patients immunized with BEC2/BCG have a male predominance compared to the BEC2/QS21 group and are slightly older. Also, 21~ of patients immunized with BEC2/BCG have either stage III with more than 4 lymph nodes positive or have stage IV disease while none of the BEC2/QS21 patients have these poor prognostic features.
Table 1. Characteristics of patients ;mml~n;zed with either BEC2/BCG or BEC2/QS21 Patient characteristicBEC2/BC~ BEC2/QS21 Male:female 9:5 3:3 Median age (years) 44.5 31.5 25 Extremity primary site 3 (21~) 2 ~33~) Axial primary site lO (71~) 4 (67~) Unknown primary site 1 (7~) 0 Stage II 1 (7%) 1 (16.5~) Stage III 12 (86~) 5 (83.5~) . 1 lymph node6 (43~) 2 (33~) 2-4 lymph nodes 4 (29~) 3 (50~) >4 lymph nodes 2 (14~) 0 Stage IV 1 (7~) 0 Median num~er o~ months2.7 (1-5.8) 3.2 (0.93-4.7) 35 ~rom surgical excision to ~irst immunization (range) CA 02243~09 l998-07-l~
W097/3463s PCT~S97/04590 Seroloqical responses A11 patients in both trials develop IgG antibodies against BEC2 (Table 2). There is no meaningful difference ~etween the median o~ range of anti-BEC2 IgG titers induced by BEC2/BCG and BEC2/QS21 (Table 2) nor in the number of immunizations required to induce anti-BEC2 IgG (data not shown). This suggests that both BCG and QS21 adjuvants are e~ually potent at augmenting anti-BEC2 antibody responses.
Table 2. Serological response rates in patients immunized _ with BEC2/BCG or BEC2 +QS21.
Y~crTM~ ANTI-BEC2 ANTIBODIES ANTI-GD3 ANTIBODI~S
% OF MEDIAN TITER ~ OF TITERS
PATIENTS (RANGE) PATIENTS
DEVELOPING DEVELOPING
ANTIBODIES
ANTIBODIES
B~C2/BCG 100 1:204800 21 1:80,1:160 1:640 (1:25600-1:1638, 400) BEC2/QS21 100 1:102400 0 (1:51200-1:204800) In order to determine whether a portion of the anti-BEC2 response represents an Ab3 response, immune sera showing high titers of anti-BEC2 IgG is tested for binding MPC11, an isotype-matched mouse MAb (IgG2) in which 75~ of the framework se~uences are identical to the BEC2 framework sequences ~unpublished). Preferential binding to BEC2, as demonstrated either by higher titer or higher absorbance readings, is observed in 11/14 (79~) patients immunized with BEC2/BCG and 5/6 (83~ of patients immunized with BEC2/QS21 implying that the majority of patients, immunization induces BEC2-specific IgG antibodies. Data from a representative patient (patient 13 immlln;zed with BEC2/BCG) is shown in CA 02243~09 l99X-07-1~
WO 97t34635 PCT/US97/04590 Figure lA.
In order to compare the relative specificity for BEC2 versus MPC11 for each treatment group, the absorbance readings for BEC2 binding are compared to the reading~ for MPC11 binding at a serum dilution that resulted in sub-maximal binding to BEC2 (absorbance at 405nm approximately e~ual to 0.5). This standardizes the assay and ensures that the assay is carried out at a dilution corresponding to a linear portion of the titration curve for each serum sample. Figure lB shows the mean absorbance readings against BEC2 and MPC11 for patients ;m~lln; zed with BEC2/BCG or BEC2/QS21. In both treatment groups, the mean absorbance against MPC11 is lower than the reading against BEC2 and again implies that immunization induced BEC2-specific IgG. To confirm the presence of specific antibodies against BEC2 (i.e. Ab3), selected sera are exhaustively absorbed against MPC11 and then tested for binding to BEC2.
Figure lC shows the results of an experiment using the same serum specimen used in Figure lA. After absorption against MPCll, no anti-MPC11 reactivity remains but substantial reactivity against BEC2 is still observed demonstrating the presence of Ab3 antibodies.
Anti-GD3 antibodies were detected by ELISA in 3/14 (21~) patients immunized with BEC2/BCG (Table 2) while no patient immunized with BEC2/QS21 developed detectable anti-GD3 antibodies. The anti-GD3 antibodies were detected with secondary antibodies against human IgG but not IgM
suggesting they were of the IgG isotype. The titers of the anti-GD3 antibody responses are illustrated in Figure 2 and ranged ~rom 1:80 to 1:640.
- 35 Disease recurrence and overall survival Surviving patients immunized with BBC2/BCG have been followed for a median of 2.4 years (2.2 - 2.9) and 10/14 CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 patients (71~) remain alive. Of these 10 patients, ~ never recurred, 1 recurred with a solitary metastasis that has been resected, and 1 patient remains alive with metastatic disease. Figure 3 shows that the overall survival rate plateaued suggesting that the disease-~ree status of the remaining patients may be durable. Two of the three patients who developed anti-GD3 antibodies, including one patient who had stage IV disease, remain free of disease.
The patients immunized with BEC2/QS21 have been followed for a median of 2 years and of the six patients ~mml1n;zed, 3 remain free of disease.
Toxicity All patients immunized with BEC2/BCG experienced grade III
toxicity at the sites of immunization characterized by in~lammation, ulceration, and oozing. In one patient, the third immunization had to be delayed for 1 month to allow the previous vaccine sites to heal. All sites healed with m; nim~l wound care and there were no instances of BCGosis.
Eight patients experienced grade II fever, 3 noted grade II
fatigue or malaise, and one patient had a transient grade II
elevation in glucose. Grade I toxicities included elevated liver function tests (6 patients), hyperglycemia (3 patients), fatigue (3 patients), fever (1 patient), and hypocalcemia (1 patient).
All patients immunized with BEC2/QS21 also experienced inflammation at the sites of injection although only of grade I~ severity and of shorter duration; there was no ulc~ ation or oozing. Two of the patients also noted grade II fever. Grade I toxicities included fatigue (4 pati.ents), hyperglycemia (2 patients), neutropenia (2 patients), and diarrhea (l patient).
Overall, immunizations are well-tolerated and no patient is removed from study because of adverse reactions.
CA 02243~09 1998-07-1~
W097/3463s PCT~S97/04590 HLA class II oliqotypinq As an exogenous antigen, BEC2 may be taken up by antigen presenting cells, processed to peptides, and helper T cell epitopes presented by HLA class II molecules. In this way, 5 BEC2 may provide T cell help for the B cell response against GD3. Therefore, applicants are interested in knowing the HLA class II type of the patients to determine whether there is any correlation between HLA class II type and immune response to BEC2 or overall survival.
Table 3. HLA class II genotyping of melanoma patients immunized with BEC2 /BCG
Oligotyping Serologically-de~ined all~l~s' Pts DR allele~ DQ ~llele~ DR DQ
1 5 #
IBl*0701 Bl*1104B4*0101 B3*02 Bl*0201 Bl*0301 7, 11 w53, 2.7 w52 3Bl*0301 Bl*1103B3*0101 B3*02 Bl*0201 Bl*0301 3, 11,w52 2,7 4Bl*07Bl*0801B4*0101 Bl*0201 Bl*0402 7, 8,wS3 2,4 5Bl*0301 Bl*1103B3*0101 B3*02 Bl*0201 Bl*0301 3, 11,w52 2,7 2 0 6Bl*ISOI Bl*1104BS*OIOI B3*02 Bl*0602 Bl*0301 15, 11,w52 1,7 7Bl*1302 Bl*1306B3*02 B3*0301 Bl*0603 Bl*0605 13,wS2 8Bl*ISOI Bl*04B5*0101 B4*0101 Bl*0602 Bl*0605 15, 4,wS3 10Bl*0301 Bl*0402B3*0101 B4*0101 Bl*0201 Bl*0302 3, 4,w52, 2,3 w53 Il Bl*0102Bl*1104B3*02 BI~OS01 Bl*0301 1, 1I,w52 7 12 Bl*14 Bl*04 B3*02 B4*0101 Bl*0503 Bl*0301 14, 4, w52, 1,7 w53 13 Bl*0701Bl*1104B3*02 B4*01 Bl*0201 B1*0301 7, 11,w52, 2,7 w53 14 Bl*0301 B3*0101 Bl*0201 3 2 CA 02243~09 l998-07-l~
W097/34635 PCT~S~7/04S90 Class II oligotyping is performed in 12 of the 14 patients immunized with BEC2/BCG (Table 3); samples are not available in the remaining two BEC2/BCG patients. Oligotyping is not carried out in the BEC2/QS21 patients due to the small number of these patients. There is no apparent associ.ation between HLA class II genotype and induction of anti-GD3 antibodies, induction of anti-BEC2 antibodie~, or survival.
HLA class II oligotyping is performed as described in Materials and Methods. DNA is extracted from PBMC except in patient ~1 in whom DNA is extracted from hair bulbs. DNA is not available from patients 2 or 9. ~Serological designations are derived from the oligotyping data and are not directly determined.
The corresponding serologically-defined class II allele distribution among the patients were compared with the distribution published for the Caucasian population in the United States in general (Rieger, et al). This re~erence group (N=232) was used since it best reflected the group of twelve melanoma patients. While there are no apparent differences at the DR locus, there is an apparent decrease in DQ1 frequency (43.7~ vs. 25~) with a corresponding increase in the frequency of DQ2 (22.9~ vs. 33.3~) and DQ7 (16.3~ vs. 33.3~) among the melanoma patients. However, a formal statistical test is not performed due to the small sample size. These data are consistent with the hypothesis that certain HLA class II alleles, or the loss of certain alleles, may be associated with an increased risk o~
melanoma.
Ex~erimental Di 8CU8 8 ion These clinical trials extend the previous observations in melanoma patients immunized with BEC2 alone. In a previous study in which patients were immunized with ~EC2 administered subcutaneously but without an immune adjuvant, CA 02243~09 l998-07-l~
W097/34635 PCT~S97/~4590 only 55~ of patients developed anti-BEC2 antibodies and only 1/20 developed detectable anti-GD3 antibodies (Chapman, et al., 1994). In the current studies in which patients are immunized with the same dose and schedule of BEC2 mixed with either BCG or QS21 adjuvant, all patients develop anti-BEC2 antibodies and at a significantly higher titer than seen in patients immunized without adjuvant. This difference is statistically significant (p=O.O01, Fisher's exact test).
In addition, 3/14 patients immunized with BEC2/BCG develop anti-GD3 antibodies while none of the 6 patients immunized with BEC2/QS21 develop anti-GD3 antibodies. Thus, both BCG
and QS21 are potent adjuvants that enhance the anti-BEC2 antibody response but anti-GD3 responses are observed only in patients receiving BEC2/BCG.
This contrasts somewhat with what was observed previously with the MELIMMUNE anti-idiotypic MAb vaccine which mimics the high molecular weight-melanoma associated antigen (HMW-MAA). In that stuZy, QS21 was superior to BCG in inducing anti-MELIMMUNE antibodies (Livingston, et al., 1995).
While antibody responses to carbohydrate antigens, such as GD3, are generally of the IgM isotype, it is interesting to note that the anti-GD3 responses detected after immunization with BEC2 are IgG. This is consistent with what we have observed previously in rabbits immunized with BEC2 (Chapman, et al., 1991) and may be due to the fact that as a protein that mimics GD3, BEC2 is potentially capable of providing T
cell help necessary for isotype class switching.
An intriguing observation is that after a median follow-up of 2.4 years, 10/14 (71%) patients immunized with BEC2/BCG
remain alive and 9/14 (64~) free of disease. This compares favorably with the survival of stage III melanoma patients ~ 35 in general (Livingston, et al., 1995) and stage III patients rendered free of disease after surgery (Balch, et al., 1992), and although the follow-up is relatively short, both CA 02243~09 l998-07-l~
W097/3463~ PCT~97/04590 previous observations (Livingston, et al., 1995) and the fact that no recurrences have been observed for the past 19 months suggest that most of the recurrences have already occurred. It is interesting to note that many long-term survivors did not have detectable serum antibodies against GD3 after BEC2 immunization. This may be due to inadequate sensitivity o~ the anti-GD3 serological assays;
alternatively, it may be that non-antibody mechanism~ play a role in the protective effect observed. Randomized ~Drepper, et al.,1993) and non-randomized trials (Parkinson, et al., 1992) using BCG alone in melanoma patients have not demonstrated an effect of BCG on disease-free survival, and so it is considered unlikely that BCG alone is responsible ~or the effect of BEC2/BCG on survival.
This disease-free survival rate in the BBC2/BCG patients contrasts with observations in patients from a previous study in which a similar population of patients were immunized with BEC2 without adjuvant (Broker, et al., L986).
In that study, none of the patients developed anti-GD3 antibodies and only 33~ of patients were alive after a median ~ollow-up of 2.8 years. These observations suggest that highly immunogenic formulations of BEC2 can induce anti-GD3 antibodies in patients and may contribute to prolonged survival.
The HLA class II type in the patients immunized with BEC2/BCG was determined and found no apparent association between class II genotype and serological response to BEC2 or to disease-free survival. A previous report found that DQB1*0301 (corresponding serologically to DQ7), was more common among melanoma patients (56~) than among a healthy control group (27~) and that DQ~1*0301 correlated with an increased risk of advanced disease (Chapman, et al., ~995).
The applicants found a similar incidence o~ DQB1*0301 among our patients (58~) and also noted that the DQ7 allele frequency among our patients was higher than the frequency .~
CA 02243~09 Isg8-07-l~
W097/34635 PCT~S97/04590 published for Caucasians in the U.S. The signi~icance of these ~indings must be interpreted with caution given the small number of patients studied, and it remains unclear whether the incidence of DQB1*0301 (DQ7) differs significantly from a carefully matched control group.
Despite this, it i6 of interest that the current study is now the second report o~ observations consistent with an increase in frequency of DQ7 among melanoma patients.
CA 02243~09 l998-07-l~
W097/34635 PCT~S~7/04590 Reference~ of the preceding section:
1. Balch, C.M., Soong, S., Shaw, H.M., Urist, M.M. and McCarthy, W.H. An analysis of prognostic factors in 8500 patients with cutaneous melanoma. In: Balch, C.M., Houghton, A.N., Milton, G.W., Sober, A.J. and Soong, S.
(eds.) Cutaneous Melanoma, Ed. 2nd. pp. 165-187.
Philadelphia: J.B. Lippincott Company, 1992.
2. Brocker, E.B., Suter, L., Czarnetzki, B.M. and Macher, E. BCG immunotherapy in stage I melanoma patients.
Does it influence prognosis determined by F~LA-DR
expression in high-risk primary tumors? Cancer Immunol Immunother, 23:155-157, 1986.
ANTI - IDIOTYPIC ANTTBOD~ VACCINES
This application is a continuation-in-part of United States Patent Application Serial No. 08/619,217, ~iled March 21, 1996, the contents of which are hereby incorporated by reference.
The invention disclosed herein was made with the support o~
the NCI Grant No. PO1-CA33049. Accordingly, the U.S.
Government has certain rights in this invention.
Throughout this application, various publications are referenced by author and date. Full citations for these publications may be ~ound listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Backqround of the }nvention GD3 ganglioside is an appealing target for immunotherapy of melanoma for several reasons. Expression of the GD3 ganglioside on normal tissue is selective and occurs at low concentrations. Expression of the GD3 ganglioside is abundant on most melanoma (Hamilton et al., 1993) and antibodies to GD3 can both block melanoma cell attachment to surfaces (Cheresh, et al., 1986; Chapman, et al., l990), an early critical step in invasion and metastasis, and can inhibit melanoma cell growth in vitro (Dippold, et al., 1984). In animal models, monoclonal antibodies (MAb) R24 against GD3 can inhibit outgrowth of melanoma tumors and treatment of metastasic melanoma patients with the anti-GD3 MAb R24 can result in ma3or tumor shrinkage (Vadhan-Raj, et al., 1988; Caufield, et al., 1990; Raymond, et al., l991;
Creekmore, et al., 1992). However, GD3 is poorly immunogenic, and attempts to immunize patients against GD3 using GD3~ cells, puri~ie~ GD3, or GD3 conjugates have been CA 02243~09 1998-07-1~
unsucces6~ul (Tai, et al., 1985; Livingston, et al., 1989).
United States Patent Number 5,529,922, issued June 25, 1996, the content o~ which is incorporated into this application, discloses an anti-idiotypic monoclonal antibody, BEC2 made against the anti-GD3 monoclonal antibody R24 and a method to produce the same. The anti-idiotypic monoclonal antibody, BEC2 carries the internal image of GD3 and then mimics GD3.
Such monoclonal antibody is a valuable immunological reagent. Patients were immunized with BEC2 administered subcutaneously without an immune adjuvant; only 55% o~
patients developed anti-BEC2 antibodies and only 1/20 developed detectable anti-GD3 antibodies.
Potent immunological adjuvants, as QS21 and BCG, have been used to enhance the immune response to ganglioside and to anti-idiotypic MAb-(Mittelman, et al., 1992). QS21 is a saponin extracted from the bark of the South American soap bark tree Quillaja saponaria Molina with significant adjuvant activity ~Kensil, et al., 1991; Wu, et al., 1992;
Newman, et al., 1992; White, et al., 1991). Because of its wide spread use, high adjuvant effect and low toxicity QS21 is best selected. BCG is an attenuated strain o~
Mycobacterium bovis and is a potent immunological adjuvant that has been used to enhance the immune response to gangliosides (Livingston, et al., 1994) and to anti-idiotypic MAb (Mittelman, et al., 1992).
CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 Summary o~ the Invention The present invention provides a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle that enhance the production of antibodies against GD3.
The present invention also provides a method for ~nh~ncing and stimulating production of antibodies against GD3 in a melanoma subject which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle.
The present invention further provides a method for treating melanomas patients which compri~es administering to the subJect a vacclne comprislng an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle.
The present invention further provides a method for preventing recurrence of melanomas which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle.
In one embodiment of the invention, the invention provides a method for increasing the survival rate of small cell lung ~ cancer patients which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and a pharmaceutically acceptable vehicle.
CA 02243~09 l998-07-l~
W097/34635 PCT~Ss7/04590 Brief Description o~ the Fiqures Fiqure 1:
A. Reactivity of immune serum from patie~t 13 immunized with BEC2/BCG against BEC2 (-) and MPC11 (-). After 5 immunizations immune serum is diluted as show~ and tested for binding to BEC2 and MPC11 as described in the Experimental section.
B. Mean reactivity of immune sera against BEC2 (solid bars) or MPCll (open bars) in patients immunized with BEC2/BCG (N=14) or BEC2/QS21 (N=6). Each serum sample is tested at a dilution that resulted in absorbance readings of approximatel~ 0.5 against BEC2-coated plates. Error bars represent standard deviation of the mean.
C. Binding of pre-absorbed serum ~rom patient 13 immunized with BEC2/BCG against BEC2 (-) and MPC11 (-). The same serum as shown in Figure lA (diluted to l:lOO,OO0) is exhaustively absorbed against MPC11-coated agarose beads prior to performing the assay.
Fioure 2:
A. Anti-GD3 antibody titers induced by BEC2/BCG in patient 1. Serological assays were performed as described in the Experimental section. Arrows indicate immunization with BEC2/BCG. In this patient, anti-GD3 reactivity was detected only with alkaline phosphatase-conjugated second antibody against human IgG.
B. Anti-GD3 antibody titers induced by BEC2/BCG in CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 patient 3. Serological assays were per~ormed as described in the Experimental section. Arrows indicate immunization with BEC2/BCG. In this patient, anti-GD3 reactivity was detected only with alkaline phosphatase-conjugated second antibody against human IgG.
C. Anti-GD3 antibody titers induced by BEC2/BCG in patient 13. Serological assays were performed as described in the Experimental section. Arrows indicate immunization with BEC2/BCG. In this patient, anti-GD3 reactivity was detected only with alkaline phosphatase-conjugated second antibody against human IgG.
Figure 3: Overall survival o~ patients immunized with BEC2/BCG (Kaplan-Meier Curve).
Figure 4: Overall survival of SCLC patients immunized with BEC2/BCG ~Kaplan-Meier Curve).
CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 Detailed DescriPtion of the Invention The present invention provides a vaccine comprising an anti-idiotypic monoclonal antibody designated BEC2, an effective amount of an appropriate adjuvant and a pharmaceutically acceptable vehicle. This vaccine is useful for stimulating or enhancing in a subject to which the vaccine is administered, production of antibodies against GD3 ganglioside.
As u_ed herein, the term "anti-idiotypic monoclonal antibody designated BEC2-1 includes the mouse BEC2 antibody, chimeric antibody of the mouse BEC2 antibody, single chain antibody of BEC2 and functional fragments of these antibodies.
Preferably, the chimeric antibody is a humanized BEC2 antibody wherein the framework of the immunoglobin molecule is hllm~n and only the Complementarity Determining Region is from mouse. In addition, the antibody may be a single chain antibody. Functional fragments may be the Fab or Fab' fragments. The method to make the functional fragments, chimeric antibodies or single chain antibody of BE~2 are well-known in the art.
In an embodiment, the effective amount of BEC2 ranges from about O. 5 mg to 25 mg. In another embodiment, the effective amount of BEC 2 is 2.5 mg.
In a separate embodiment, the appropriate adjuvant is BCG.
In a further embodiment, BCG is administered in attenuated doses.
In a preferred em~odiment, the vaccine is administered in at least one site on definite time intervals. In a further embodiment, the vaccine is administered in one 03~ more sites. The administration may be effected intradermally, su~cutaneously, parenterally, or intravenously.
CA 02243jO9 l998-07-lj W097/34635 PCT~S97/04590 The present invention also provides a method for enhancing and stimulating production of antibodies against GD3 in a melanoma subject which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of an appropriate adjuvant and a pharmaceutically acceptable vehicle.
The present invention further provides a method for treating melanoma in a subject which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of an appropriate adjuvant and an pharmaceutically acceptable vehicle.
The present invention further provides a method for preventing recurrence of cancer which comprises administering to a melanoma subject a vaccine comprising an e~fective amount of the anti-idiotypic monoclonal antibody designated BEC2, an e~fective amount of an appropriate adjuvant and an acceptable pharmaceutical vehicle.
In one embodiment of the invention, the invention provides a method for increasing the survival rate of small cell lung cancer patients which comprises administering to the subject a vaccine comprising an effective amount of the anti-idiotypic monoclonal antibody designated BEC2, an effective amount of the appropriate adjuvant and an pharmaceutically acceptable vehicle.
In another em~odiment of the invention, the invention provides a method for increasing the survival rate o~ small cell lung cancer patients which comprises administering to the subject a vaccine comprising an effectlve amount of the anti-idiotypic monoclonal antibody designated BEC2, an e~fective amount of the appropriate adjuvant and an pharmaceutically acceptable vehicle in combination with a CA 02243~09 l998-07-l~
W097/3463S PCT~97/04590 treatment with a chemotherapeutic agent.
In another embodiment of the invention, the appropriate adjuvant is BCG.
In another embodiment of the invention, the ef~ective amount of the anti-idiotypic monoclonal antibody, BEC2, ranges from about 0.5 mg to about 25 mg.
In another embodiment of the invention, the effective amount of the anti-idiotypic monoclonal antibody, BEC2, is about 2.5 mg.
Different effective amounts of the anti-idiotypic monoclonal antibody, BEC2 thereof may be used according to this invention. A person ordinary skilled in the art can perform simple titration experiments to determine what effective amount is required for effective immunization. An example of such titration experiment is to inject different amounts of the anti-idiotypic monoclonal antibody, BEC2, from 0.5 mg to 32 mg, to make a seroconversion and then ~m; ne the immune response.
In another embodiment of the invention, the adjuvant is BCG
and an effective amount of BCG is about 107 Colony Forming Units (CFU).
Different effective amounts of the appropriate adjuvant thereof may be used according to this invention. A person ordinary skilled in the art can perform simple titration experiments to determine what effective amount is required for effective immunization. An example of such titration experiment is to inject different amounts of the appropriate adjuvant and then examine the immune response.
In another embodiment of the invention, the vaccine comprising 2.5mg of BEC2 and attenuated doses of BCG is CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 injected in weeks 0,2,4,6 and 10.
In another embodiment of the invention, subsequent doses of BCG are 3-fold attenuated.
In another embodiment of the invention, the vaccine is injected intradermally, subcutaneously or intravenously.
Pharmaceutically acceptable carriers are well known to those skilled in the art. Such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the speci~ic experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follow thereafter.
CA 02243~09 l998-07-l~
W097/3463S PCT~S97/0459 Ex~erimental Detail~:
First serie~ of ex~eriment~:
Material and Methods Patients selection Se~uential clinical trials in which melanoma patients free of disease after complete surgical resection but at high risk for recurrence are immunized with BEC2 mixed with BCG
(BEC2/BCG) using 2.5 mg of BEC2. Patients free of disease afte_ resection of AJCC stage II are also eligible if the primary tumor was 24mm deep. Other eligibility re~uirements are: normal WBC and differential count, no previous chemotherapy, immunotherapy, or radiation therapy within the previous month, and no history of other malignancies within the past 5 years. Patients are excluded if they have significant underlying medical problems (e.g. NYHA class III
or IV cardiac disease, active infections requiring antibiotics, or active bleeding), required antihistamines, steroids, or non-steroidal anti-inflammatory drugs, have an immunodeficiency or are asplenic, have a history of metastases to the CNS, or are pregnant or lactating.
Vaccine components BEC2 is purified from virus-free lipoprotein-free cell culture supernatants by Celltech, Ltd (Slough, Bngland) using Protein A affinity chromatography and anion-exchange chromatography. The final product is >95~ pure as determined by SDS-polyacrylamide gel electrophoresis. BEC2 is supplied in sterile phosphate buffered saline at concentration of 2.5 mg/ml in single-dose vials.
BCG (TheraCys~) is purchased from Connaught Laboratories (Swiftwater, PA) in vials containing 3.4 x 108 colony-forming units (CFU)/vial in a ~reeze-dried form. Immediately before administration, BCG is reconstituted with 3.4 ml of sterile diluent and then further diluted in normal saline if CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 necessary. Vaccine is prepared by mixing 1 ml o~ BEC2 (2.5 mg) with O.l ml of the appropriate dilution of BCG
immediately prior to administration.
QS21, supplied by Cambridge Bioscience, Inc. (Worcester, MA), is extracted ~rom Quillaja saponaria Molina tree bark using silica and reverse-phase chromatography as previously described (Kensil, et al., 1991). It is supplied in 2.1 mg vials as a lyophilized powder and once reconstituted with sterile phosphate buffered saline, can be stored at 4~C for not more than 14 days. Vaccine containing QS21 is prepared immediately prior to ~min;stration by mixing 1 ml of BEC2 (2.5 mg) with lO0 ~g o~ QS21. The dose o~ QS21 is selected based on a previous phase I clinical trial (Livingston, et al., 1994).
The vaccine is injected intradermally into multiple rotated sites on weeks O, 2, 4, 6, and lO; injections are given only into limbs in which the regional lymph node basin is intact.
In patients who developed anti-GD3 antibodies, a sixth immunization is offered at a time when the anti-GD3 antibody titers returned to baseline.
The initial immunization contained 2.5 mg BEC2 mixed with 1 x 107 CFU o~ BCG. In the setting of grade III local toxicity, subsequent doses o~ BCG are attenuated 3-~old (i.e. 3 x 106 CFU, 1 x 106 CFU, 3 x 105 CFU, 1 x lO~ CFU, 3 x 104 CFU, 1 x 104 CFU). Patients with a positive PPD (or a hlctory of a positive PPD), are started at an atte~uated BCG
dose of 3 x 106 CFU.
Vaccine is administered as a single subcutaneous injection using the identical schedule and dose o~ BEC2 as ~or the BEC2/BCG vaccine (wee~s O, 2, 4, 6, and lO). Each vaccine consisted o~ 2.5 mg BEC2 and 100 ~g of QS21; the QS21 dose CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 is not attenuated.
Seroloqical evaluat.ion Serum is collected pre-treatment, at the time o~ each immunization, and two weeks following each immunization and stored at -20~C. Serum is tested for the presence of B~C2 antibodies and anti-GD3 antibodies using an ELISA assay as previously described (Chapman, et al., 1994). Internal standards are run with each assay to ensure adequate and reproducible sensitivity. The specificity of the anti-BEC2 response is characterized by comparing the anti-BEC2 IgG
response to the IgG response against MPC11, an isotype-matched mouse MAb (IgG2b) with framework sequences (Ng, et al., 1993~. Ab3 antibodies are detected by their ability to bind BEC2 but not MPC11. The degree of binding of BEC2 versus MPC11 is compared by ELISA using a serum dilution that results in sub-maximal binding to BEC2 (absorbance values o~ approximately 0.5 are used). This is done ~irstly to ensure that the assay is carried out at a serum dilution corresponding to the linear portion of the titration curve for each serum sample and secondly, to st~n~dize the assay allowing the data to be analyzed according to treatment group. The presence of Ab3 antibodies is con~irmed in selected patients by exhaustively absorbing immune serum against agarose beads ~Affigel lO, Bio-Rad Laboratories, ~ercules, CA) coated with MPC11 until reactivity against MPC11 was not detected. The absorbed serum is then assayed ~or binding to BEC2.
A patient is considered to have developed anti-GD3 antibodies if at least two post-treatment sera samples reproducibly demonstrate a 4-fold or greater increase in anti-GD3 reactivity compared to the pre-treatment sample.
This strict criterion was established based on our serological studies in patients immunized with BCG alone to avoid confusion with transient, low-level, non-specific reactivity that can be induced by BCG and other adjuvants.
CA 02243509 l998-07-l5 W097/3463~ PCT~S97/04590 Attempts to characterize further the specificity of anti-GD3 responses employed mixed hemadsorption assays against GD3 allogeneic melanoma cell lines (Vadhan-Raj, et al., 1988) and immuno-thin layer chromatography ~Chapman, et al., 1994).
Ex~erimental Results:
Patient characteristics Fourteen patients are immunized with BEC2/BCG while 6 patients are immunized with BEC2/QS21. Patient characteristics are shown in Table 1. While the two groups of patients are relatively similar, the patients immunized with BEC2/BCG have a male predominance compared to the BEC2/QS21 group and are slightly older. Also, 21~ of patients immunized with BEC2/BCG have either stage III with more than 4 lymph nodes positive or have stage IV disease while none of the BEC2/QS21 patients have these poor prognostic features.
Table 1. Characteristics of patients ;mml~n;zed with either BEC2/BCG or BEC2/QS21 Patient characteristicBEC2/BC~ BEC2/QS21 Male:female 9:5 3:3 Median age (years) 44.5 31.5 25 Extremity primary site 3 (21~) 2 ~33~) Axial primary site lO (71~) 4 (67~) Unknown primary site 1 (7~) 0 Stage II 1 (7%) 1 (16.5~) Stage III 12 (86~) 5 (83.5~) . 1 lymph node6 (43~) 2 (33~) 2-4 lymph nodes 4 (29~) 3 (50~) >4 lymph nodes 2 (14~) 0 Stage IV 1 (7~) 0 Median num~er o~ months2.7 (1-5.8) 3.2 (0.93-4.7) 35 ~rom surgical excision to ~irst immunization (range) CA 02243~09 l998-07-l~
W097/3463s PCT~S97/04590 Seroloqical responses A11 patients in both trials develop IgG antibodies against BEC2 (Table 2). There is no meaningful difference ~etween the median o~ range of anti-BEC2 IgG titers induced by BEC2/BCG and BEC2/QS21 (Table 2) nor in the number of immunizations required to induce anti-BEC2 IgG (data not shown). This suggests that both BCG and QS21 adjuvants are e~ually potent at augmenting anti-BEC2 antibody responses.
Table 2. Serological response rates in patients immunized _ with BEC2/BCG or BEC2 +QS21.
Y~crTM~ ANTI-BEC2 ANTIBODIES ANTI-GD3 ANTIBODI~S
% OF MEDIAN TITER ~ OF TITERS
PATIENTS (RANGE) PATIENTS
DEVELOPING DEVELOPING
ANTIBODIES
ANTIBODIES
B~C2/BCG 100 1:204800 21 1:80,1:160 1:640 (1:25600-1:1638, 400) BEC2/QS21 100 1:102400 0 (1:51200-1:204800) In order to determine whether a portion of the anti-BEC2 response represents an Ab3 response, immune sera showing high titers of anti-BEC2 IgG is tested for binding MPC11, an isotype-matched mouse MAb (IgG2) in which 75~ of the framework se~uences are identical to the BEC2 framework sequences ~unpublished). Preferential binding to BEC2, as demonstrated either by higher titer or higher absorbance readings, is observed in 11/14 (79~) patients immunized with BEC2/BCG and 5/6 (83~ of patients immunized with BEC2/QS21 implying that the majority of patients, immunization induces BEC2-specific IgG antibodies. Data from a representative patient (patient 13 immlln;zed with BEC2/BCG) is shown in CA 02243~09 l99X-07-1~
WO 97t34635 PCT/US97/04590 Figure lA.
In order to compare the relative specificity for BEC2 versus MPC11 for each treatment group, the absorbance readings for BEC2 binding are compared to the reading~ for MPC11 binding at a serum dilution that resulted in sub-maximal binding to BEC2 (absorbance at 405nm approximately e~ual to 0.5). This standardizes the assay and ensures that the assay is carried out at a dilution corresponding to a linear portion of the titration curve for each serum sample. Figure lB shows the mean absorbance readings against BEC2 and MPC11 for patients ;m~lln; zed with BEC2/BCG or BEC2/QS21. In both treatment groups, the mean absorbance against MPC11 is lower than the reading against BEC2 and again implies that immunization induced BEC2-specific IgG. To confirm the presence of specific antibodies against BEC2 (i.e. Ab3), selected sera are exhaustively absorbed against MPC11 and then tested for binding to BEC2.
Figure lC shows the results of an experiment using the same serum specimen used in Figure lA. After absorption against MPCll, no anti-MPC11 reactivity remains but substantial reactivity against BEC2 is still observed demonstrating the presence of Ab3 antibodies.
Anti-GD3 antibodies were detected by ELISA in 3/14 (21~) patients immunized with BEC2/BCG (Table 2) while no patient immunized with BEC2/QS21 developed detectable anti-GD3 antibodies. The anti-GD3 antibodies were detected with secondary antibodies against human IgG but not IgM
suggesting they were of the IgG isotype. The titers of the anti-GD3 antibody responses are illustrated in Figure 2 and ranged ~rom 1:80 to 1:640.
- 35 Disease recurrence and overall survival Surviving patients immunized with BBC2/BCG have been followed for a median of 2.4 years (2.2 - 2.9) and 10/14 CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 patients (71~) remain alive. Of these 10 patients, ~ never recurred, 1 recurred with a solitary metastasis that has been resected, and 1 patient remains alive with metastatic disease. Figure 3 shows that the overall survival rate plateaued suggesting that the disease-~ree status of the remaining patients may be durable. Two of the three patients who developed anti-GD3 antibodies, including one patient who had stage IV disease, remain free of disease.
The patients immunized with BEC2/QS21 have been followed for a median of 2 years and of the six patients ~mml1n;zed, 3 remain free of disease.
Toxicity All patients immunized with BEC2/BCG experienced grade III
toxicity at the sites of immunization characterized by in~lammation, ulceration, and oozing. In one patient, the third immunization had to be delayed for 1 month to allow the previous vaccine sites to heal. All sites healed with m; nim~l wound care and there were no instances of BCGosis.
Eight patients experienced grade II fever, 3 noted grade II
fatigue or malaise, and one patient had a transient grade II
elevation in glucose. Grade I toxicities included elevated liver function tests (6 patients), hyperglycemia (3 patients), fatigue (3 patients), fever (1 patient), and hypocalcemia (1 patient).
All patients immunized with BEC2/QS21 also experienced inflammation at the sites of injection although only of grade I~ severity and of shorter duration; there was no ulc~ ation or oozing. Two of the patients also noted grade II fever. Grade I toxicities included fatigue (4 pati.ents), hyperglycemia (2 patients), neutropenia (2 patients), and diarrhea (l patient).
Overall, immunizations are well-tolerated and no patient is removed from study because of adverse reactions.
CA 02243~09 1998-07-1~
W097/3463s PCT~S97/04590 HLA class II oliqotypinq As an exogenous antigen, BEC2 may be taken up by antigen presenting cells, processed to peptides, and helper T cell epitopes presented by HLA class II molecules. In this way, 5 BEC2 may provide T cell help for the B cell response against GD3. Therefore, applicants are interested in knowing the HLA class II type of the patients to determine whether there is any correlation between HLA class II type and immune response to BEC2 or overall survival.
Table 3. HLA class II genotyping of melanoma patients immunized with BEC2 /BCG
Oligotyping Serologically-de~ined all~l~s' Pts DR allele~ DQ ~llele~ DR DQ
1 5 #
IBl*0701 Bl*1104B4*0101 B3*02 Bl*0201 Bl*0301 7, 11 w53, 2.7 w52 3Bl*0301 Bl*1103B3*0101 B3*02 Bl*0201 Bl*0301 3, 11,w52 2,7 4Bl*07Bl*0801B4*0101 Bl*0201 Bl*0402 7, 8,wS3 2,4 5Bl*0301 Bl*1103B3*0101 B3*02 Bl*0201 Bl*0301 3, 11,w52 2,7 2 0 6Bl*ISOI Bl*1104BS*OIOI B3*02 Bl*0602 Bl*0301 15, 11,w52 1,7 7Bl*1302 Bl*1306B3*02 B3*0301 Bl*0603 Bl*0605 13,wS2 8Bl*ISOI Bl*04B5*0101 B4*0101 Bl*0602 Bl*0605 15, 4,wS3 10Bl*0301 Bl*0402B3*0101 B4*0101 Bl*0201 Bl*0302 3, 4,w52, 2,3 w53 Il Bl*0102Bl*1104B3*02 BI~OS01 Bl*0301 1, 1I,w52 7 12 Bl*14 Bl*04 B3*02 B4*0101 Bl*0503 Bl*0301 14, 4, w52, 1,7 w53 13 Bl*0701Bl*1104B3*02 B4*01 Bl*0201 B1*0301 7, 11,w52, 2,7 w53 14 Bl*0301 B3*0101 Bl*0201 3 2 CA 02243~09 l998-07-l~
W097/34635 PCT~S~7/04S90 Class II oligotyping is performed in 12 of the 14 patients immunized with BEC2/BCG (Table 3); samples are not available in the remaining two BEC2/BCG patients. Oligotyping is not carried out in the BEC2/QS21 patients due to the small number of these patients. There is no apparent associ.ation between HLA class II genotype and induction of anti-GD3 antibodies, induction of anti-BEC2 antibodie~, or survival.
HLA class II oligotyping is performed as described in Materials and Methods. DNA is extracted from PBMC except in patient ~1 in whom DNA is extracted from hair bulbs. DNA is not available from patients 2 or 9. ~Serological designations are derived from the oligotyping data and are not directly determined.
The corresponding serologically-defined class II allele distribution among the patients were compared with the distribution published for the Caucasian population in the United States in general (Rieger, et al). This re~erence group (N=232) was used since it best reflected the group of twelve melanoma patients. While there are no apparent differences at the DR locus, there is an apparent decrease in DQ1 frequency (43.7~ vs. 25~) with a corresponding increase in the frequency of DQ2 (22.9~ vs. 33.3~) and DQ7 (16.3~ vs. 33.3~) among the melanoma patients. However, a formal statistical test is not performed due to the small sample size. These data are consistent with the hypothesis that certain HLA class II alleles, or the loss of certain alleles, may be associated with an increased risk o~
melanoma.
Ex~erimental Di 8CU8 8 ion These clinical trials extend the previous observations in melanoma patients immunized with BEC2 alone. In a previous study in which patients were immunized with ~EC2 administered subcutaneously but without an immune adjuvant, CA 02243~09 l998-07-l~
W097/34635 PCT~S97/~4590 only 55~ of patients developed anti-BEC2 antibodies and only 1/20 developed detectable anti-GD3 antibodies (Chapman, et al., 1994). In the current studies in which patients are immunized with the same dose and schedule of BEC2 mixed with either BCG or QS21 adjuvant, all patients develop anti-BEC2 antibodies and at a significantly higher titer than seen in patients immunized without adjuvant. This difference is statistically significant (p=O.O01, Fisher's exact test).
In addition, 3/14 patients immunized with BEC2/BCG develop anti-GD3 antibodies while none of the 6 patients immunized with BEC2/QS21 develop anti-GD3 antibodies. Thus, both BCG
and QS21 are potent adjuvants that enhance the anti-BEC2 antibody response but anti-GD3 responses are observed only in patients receiving BEC2/BCG.
This contrasts somewhat with what was observed previously with the MELIMMUNE anti-idiotypic MAb vaccine which mimics the high molecular weight-melanoma associated antigen (HMW-MAA). In that stuZy, QS21 was superior to BCG in inducing anti-MELIMMUNE antibodies (Livingston, et al., 1995).
While antibody responses to carbohydrate antigens, such as GD3, are generally of the IgM isotype, it is interesting to note that the anti-GD3 responses detected after immunization with BEC2 are IgG. This is consistent with what we have observed previously in rabbits immunized with BEC2 (Chapman, et al., 1991) and may be due to the fact that as a protein that mimics GD3, BEC2 is potentially capable of providing T
cell help necessary for isotype class switching.
An intriguing observation is that after a median follow-up of 2.4 years, 10/14 (71%) patients immunized with BEC2/BCG
remain alive and 9/14 (64~) free of disease. This compares favorably with the survival of stage III melanoma patients ~ 35 in general (Livingston, et al., 1995) and stage III patients rendered free of disease after surgery (Balch, et al., 1992), and although the follow-up is relatively short, both CA 02243~09 l998-07-l~
W097/3463~ PCT~97/04590 previous observations (Livingston, et al., 1995) and the fact that no recurrences have been observed for the past 19 months suggest that most of the recurrences have already occurred. It is interesting to note that many long-term survivors did not have detectable serum antibodies against GD3 after BEC2 immunization. This may be due to inadequate sensitivity o~ the anti-GD3 serological assays;
alternatively, it may be that non-antibody mechanism~ play a role in the protective effect observed. Randomized ~Drepper, et al.,1993) and non-randomized trials (Parkinson, et al., 1992) using BCG alone in melanoma patients have not demonstrated an effect of BCG on disease-free survival, and so it is considered unlikely that BCG alone is responsible ~or the effect of BEC2/BCG on survival.
This disease-free survival rate in the BBC2/BCG patients contrasts with observations in patients from a previous study in which a similar population of patients were immunized with BEC2 without adjuvant (Broker, et al., L986).
In that study, none of the patients developed anti-GD3 antibodies and only 33~ of patients were alive after a median ~ollow-up of 2.8 years. These observations suggest that highly immunogenic formulations of BEC2 can induce anti-GD3 antibodies in patients and may contribute to prolonged survival.
The HLA class II type in the patients immunized with BEC2/BCG was determined and found no apparent association between class II genotype and serological response to BEC2 or to disease-free survival. A previous report found that DQB1*0301 (corresponding serologically to DQ7), was more common among melanoma patients (56~) than among a healthy control group (27~) and that DQ~1*0301 correlated with an increased risk of advanced disease (Chapman, et al., ~995).
The applicants found a similar incidence o~ DQB1*0301 among our patients (58~) and also noted that the DQ7 allele frequency among our patients was higher than the frequency .~
CA 02243~09 Isg8-07-l~
W097/34635 PCT~S97/04590 published for Caucasians in the U.S. The signi~icance of these ~indings must be interpreted with caution given the small number of patients studied, and it remains unclear whether the incidence of DQB1*0301 (DQ7) differs significantly from a carefully matched control group.
Despite this, it i6 of interest that the current study is now the second report o~ observations consistent with an increase in frequency of DQ7 among melanoma patients.
CA 02243~09 l998-07-l~
W097/34635 PCT~S~7/04590 Reference~ of the preceding section:
1. Balch, C.M., Soong, S., Shaw, H.M., Urist, M.M. and McCarthy, W.H. An analysis of prognostic factors in 8500 patients with cutaneous melanoma. In: Balch, C.M., Houghton, A.N., Milton, G.W., Sober, A.J. and Soong, S.
(eds.) Cutaneous Melanoma, Ed. 2nd. pp. 165-187.
Philadelphia: J.B. Lippincott Company, 1992.
2. Brocker, E.B., Suter, L., Czarnetzki, B.M. and Macher, E. BCG immunotherapy in stage I melanoma patients.
Does it influence prognosis determined by F~LA-DR
expression in high-risk primary tumors? Cancer Immunol Immunother, 23:155-157, 1986.
3. Caulfield, J., Barna, B., Murthy, S., Tubbs, R., Sergi, J., Mendendorp, S. and Bukowski, R.M. Phase Ia-Ib trial of an anti-GD3 monoclonal antibody in combination with inter~eron-~ in patients with malignant melanoma. J Bio Response Mod, 9:319-328, 1990.
4. Chapman, P.B., Lonberg, M. and Houghton, A.N. Light chain variants of an IgG3 anti-GD3 monoclonal antibody and the relationship between avidity, effector functions, tumor targeting, and antitumor activity.
Cancer Res. 50:1503-1509, 1990.
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5. Chapman, P.B. and Houghton, A.N. Induction of IgG
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6. Chapman, P.B., Livingston, PØ, Morrison, M.E., Williams, L. and Houghton, A.N. Immunization of melanoma patients with anti-idiotypic monoclonal antibody BEC2 which mimics GD3 ganglioside: Pilot trials using no immunological adjuvant. Vaccine Res, 3:~-69, 1994.
CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 7. Chapman, P.B., McCa~ery, M., Zuklys, K., Giorgio, N.
and Houghton, A.N. Immunization with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: Superiority o~ the intravenous route.
Proc Am Assoc Cancer Res, 36:4931995.(Abstract) 8. Cheresh, D.A., Pierschbacher, M.D., Herzig, M.A. and Mujoo, K. Disialogangliosides GD2 and GD3 are involved in the attachment o~ human melanoma and neuroblastoma cells to extracellular matrix. J.Cell Biol.
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CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 7. Chapman, P.B., McCa~ery, M., Zuklys, K., Giorgio, N.
and Houghton, A.N. Immunization with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: Superiority o~ the intravenous route.
Proc Am Assoc Cancer Res, 36:4931995.(Abstract) 8. Cheresh, D.A., Pierschbacher, M.D., Herzig, M.A. and Mujoo, K. Disialogangliosides GD2 and GD3 are involved in the attachment o~ human melanoma and neuroblastoma cells to extracellular matrix. J.Cell Biol.
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9. Creekmore, S., Urba, W., Koop, W., Ewel, C., Hecht, T., Smith, I., Janik, J., Steis, R., Fenton, R., Sharfman, W., Conlon, K., Sznol, M., Holmlund, J., Curti, B., Gause, B., Houck, M., Beveridge, J., Jones, M. and Longo, D. Phase IB/II trial o~ R24 antibody and interleukin-2 (IL2) in melanoma. Proc Am Soc Clin Oncol, 11:3451992. (Abstract) 10. Dippold, W.G., Knuth, A. and Meyer zum Buschen~elde, K.
Inhibition o~ human melanoma cell growth in vitro by monoclonal anti-GD3-ganglioside antibody. Cancer Res.
44:806-810, 1984.
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11. Drepper, H., Bieb, B., Ho~herr, B., Hundeiker, M., Lippold, A., Otto, F., Padberg, G., Peters, A. and Wiebelt, H. The prognosis of patients with stage III
melanoma. Prospective long-term study o~ 286 patients o~ the Fachklinik Horneide. Cancer, 71:1239-1246, 1993.
melanoma. Prospective long-term study o~ 286 patients o~ the Fachklinik Horneide. Cancer, 71:1239-1246, 1993.
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CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 13. H~A l99l. Proceedings o~ the Eleven~h International Histocompati~llity Workshop and Con~erence, pp.
1073-1074. Oxford: Ox~ord University Press, 1992.
CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 13. H~A l99l. Proceedings o~ the Eleven~h International Histocompati~llity Workshop and Con~erence, pp.
1073-1074. Oxford: Ox~ord University Press, 1992.
14. Kabat, E., Wu, T., Reid-Miller, M., Perry, H. And Gottesmann, K. Sequences o~ Protein o~ Immunological Interest, U.S. Dept. O~ Health and Human Services, U.S.
Govt. Printing O~ice, 1987.
Govt. Printing O~ice, 1987.
15. Kensil, C.R., Patel, U., Lennick, M. and Marciani, D.
Separation and characterization o~ saponins with adjuvant activity ~rom Quillaja saponaria Molina cortex. J.Immunol. 146:431-437, 1991.
Separation and characterization o~ saponins with adjuvant activity ~rom Quillaja saponaria Molina cortex. J.Immunol. 146:431-437, 1991.
16. Kensil, C.R., Barrett, C., Kushner, N., Beltz, G., Storey, J., Patel, V., Recchia, J., Aubert, A. and Marciani, D. Development of a genetically engi~eered vaccine against ~eline leukemia virus in~ection.
J.Am.Vet.Med.Assoc. 199:1423-1427, 1991.
J.Am.Vet.Med.Assoc. 199:1423-1427, 1991.
17. Lee, J.E., Reveille, J.D., Ross, M.I. and Platsouca~, C.D. HLA-DQB1*0301 association with increased cutaneous melanoma risk. Int.J.Cancer. 59:510-513, 1994.
18. Livingston, P.O., Ritter, G., Oettgen, H.F. and Old, L.J. Immunization o~ melanoma patients with puri~ied gangliosides. In: Oettgen, H.F. (ed.) Gangliosides and Cancer, pp. 293-300. New York: VCH Publishers, 1989.
19. Livingston, P.O., Adluri, S., Helling, F., Yao, T.J., Kensil, C.R., Newman, M.J. and Marciani, D. Phase 1 trial o~ immunological adjuvant QS-21 with a GM2 ganglioside-keyhole limpet haemocyanin conjugate vaccine in patients with malignant melanoma. Vaccine.
35- 12:1275-1280, 1994.
35- 12:1275-1280, 1994.
20. ~ivingston, P.O., Wong, G.Y.C., Adluri, S., Tao, Y., CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04590 Padavan, M., Parente, R., Hanlon, C., Jones Calves, M., Helling, F., Ritter, G., Oettgen, H.F. and Old, L.J.
Improved survival in stage III melanoma patients with GM2 antibodies: A randomized trial of adjuvant vaccination with GM2 ganglioside. J.Clin.Oncol.
12:1036-1044, 1994.
W097/34635 PCT~S97/04590 Padavan, M., Parente, R., Hanlon, C., Jones Calves, M., Helling, F., Ritter, G., Oettgen, H.F. and Old, L.J.
Improved survival in stage III melanoma patients with GM2 antibodies: A randomized trial of adjuvant vaccination with GM2 ganglioside. J.Clin.Oncol.
12:1036-1044, 1994.
21. Livingston, P.O., Adluri, S., Zhang, S., Chapman, P., Raychaudhuri, S. and Merrit, J.A. Impact o~
immunological adjuvants and administration route on HAMA response after immunization with murine monoclonal anti~ody MELIMMUNE-1 in melanoma patients. Vaccine Res, 4:87-94, 1995.
immunological adjuvants and administration route on HAMA response after immunization with murine monoclonal anti~ody MELIMMUNE-1 in melanoma patients. Vaccine Res, 4:87-94, 1995.
22. Mittelman, A., Chen, Z.J., Yang, H., Wong, G.Y. and Ferrone, S. ~uman high molecular weight melanoma-associated antigen ~HMW-MAA) mimicry ~y mouse anti-idiotypic monoclonal antibody MK2-23: Induction of humoral anti-HMW-MAA immunity and prolongation o~
2Q survival in patients with stage IV melanoma.
Proc.Natl.Acad.Sci.,USA, 89:466-470, 1992.
2Q survival in patients with stage IV melanoma.
Proc.Natl.Acad.Sci.,USA, 89:466-470, 1992.
23. Newman, M.J., Wu, J.Y., Coughlin, R.T., Murphy, C.I., Seals, J.R., Wyand, M.S. and Kensil, C.R.
Immunogenicity and toxicity testing o~ an experimental HIV-l vaccine in nonhllm~n primates. AIDS
Res.Hum.Retroviruses, 8:1413-1418, 1992.
Immunogenicity and toxicity testing o~ an experimental HIV-l vaccine in nonhllm~n primates. AIDS
Res.Hum.Retroviruses, 8:1413-1418, 1992.
24. Ng, J., Hurley, C.K., Baxter-Lowe, L.A., Chopek, M., Coppo, P.A., Hegland, J., Kukuruga, D., Monos, D., ~osner, G., Schmeckpeper, B., Yang, S.Y., Dupont, B.
and Hartzman, B.J. Large-scale oligonucleotide typing ~or HLA-DRBl/3/4 and HLA-DQB1 is highly accurate and reliable. Tissue.Antigens. 42:473-479, 1993.
and Hartzman, B.J. Large-scale oligonucleotide typing ~or HLA-DRBl/3/4 and HLA-DQB1 is highly accurate and reliable. Tissue.Antigens. 42:473-479, 1993.
25. Parkinson, D.R., Houghton, A.N., Hersey, P. and Borden, E.C. Biologic Therapy o~ Melanoma. In: Balch, C.M., CA 02243~09 l998-07-l~
W097/34635 PCT~S~7/04590 Xoughton, A.N., Milton, G.W., Sober, A.J. and Soong, S.
(eds.) Cutaneous Melanoma, Ed. 2nd. pp. 522-541.
Philadelphia: J.B. Lippincott Company, 1992.
26. Raymond, J., Kirkwood, J., Vloc~, D., Rabkin, M., Day, R., Whiteside, T., Herberman, R., Mascari, R. and Simon, B. A phase Ib trial of murine monoclonal antibody R24 (anti-GD3) in metastatic melanoma.
Proc.Am.Soc.Clin.Oncol. 10: 2981991.(Abstract) 27. Rieger, R., Michaelis, A. and Green, M.M. Glos6ary of Ge~etics, Classical and Molecular, Ed.5th, Berlin:
Springer-Verlag, 1991.
W097/34635 PCT~S~7/04590 Xoughton, A.N., Milton, G.W., Sober, A.J. and Soong, S.
(eds.) Cutaneous Melanoma, Ed. 2nd. pp. 522-541.
Philadelphia: J.B. Lippincott Company, 1992.
26. Raymond, J., Kirkwood, J., Vloc~, D., Rabkin, M., Day, R., Whiteside, T., Herberman, R., Mascari, R. and Simon, B. A phase Ib trial of murine monoclonal antibody R24 (anti-GD3) in metastatic melanoma.
Proc.Am.Soc.Clin.Oncol. 10: 2981991.(Abstract) 27. Rieger, R., Michaelis, A. and Green, M.M. Glos6ary of Ge~etics, Classical and Molecular, Ed.5th, Berlin:
Springer-Verlag, 1991.
28. Tai, T., Cahan, L.D., Tsuchida, T., Saxton, R.E., Irie, R.F. and Morton, D.L. Immunogenicity of melanoma-associated gangliosides in cancer patients.
Int.J.Cancer, 35:607-612, 1985.
Int.J.Cancer, 35:607-612, 1985.
29. Vadhan-Raj, S., Cordon-Cardo, C., Carswell, E.A., Mintzer, D., Dantis, L., Duteau, C., Templeton, M.A., Oettgen, H.F., Old, L.J. and Houghton, A.N. Phase I
trial of a mouse monoclonal antibody against GD3 ganglioside in patients with melanoma: Induction of inflammatory responses at tumor sites. J.Clin.Oncol.
6:1636-1648, 1988.
trial of a mouse monoclonal antibody against GD3 ganglioside in patients with melanoma: Induction of inflammatory responses at tumor sites. J.Clin.Oncol.
6:1636-1648, 1988.
30. White, A.C., Cloutier, P. and Coughlin, R.T. A puri~ied saponin acts as an adjuvant for a T-independent antigen. Adv.Exp.Med.Biol. 303:207-210, 1991.
31. Wu, J.Y., Gardner, B.H., Murphy, C.I., Seals, J.R., Kensil, C.R., Recchia, J., Beltz, G.A., Newman, G.W.
and Newman, M.J. Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-l vaccine. J.Immunol. 148:1519-1525, 1992.
CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04S90 Second series of experiment~:
Since the development of combination therapy over 2Q years ago, no chemotherapeutic intervention has substantially prolonged the survival of small cell lung cancer (SCLC) patients from a median survival of 7-16 months.
Material and Method~:
Patients selection Patients who have a major response to initial therapy are injected intradermally on weeks 0, 2,4,6 and 10 with 2.5mg of BEC2 and 2x107 CFU of BCG. The dose of BCG is attenuated for skin toxicity superior or equal to 3. Patients characteristics are a median age of 63 years old, a median KPS of 80~, an elevated LDH2 in two out six patients and hyponatremia in two out six patients. (Table 4) Of 8 SCLC patients, 6 patients are evaluable for response with a median follow up of 36 months (range 16-43 months).
(table 4) Vaccine comPonents BEC2 is purified from virus-free lipoprotein-free cell culture supernatants by Celltech, Ltd (Slough, England) using Protein A affinity chromatography and anion-exchange chromatography. The final product is ~95~ pure as determined by SDS-polyacrylamide gel electrophoresis. BEC2 is supplied in sterile phosphate buffered saline at concentration of 2.5 mg/ml in single-dose vials.
BCG (TheraCys~) is purchased from Connaught Laboratories (Swiftwater, PA) in vials containing 3.4 x 108 colony-forming units (CFU)/vial in a freeze-dried form. Immediately before administration, BCG is reconstltuted with 3.4 ml of sterile diluent and then further diluted in normal saline if necessary. Vaccine is prepared by mixing 1 ml of BEC2 (2.5 CA 02243509 l998-07-l5 W097/34635 . PCT~S97/04590 mg) with 0.1 ml of the appropriate dilution of BCG
immediately prior to administration.
Table 4. Immunization of small cell lung cancer patients with BEC2 and BCG
Date o~ Dx Complete Rx Date BEC2 Last FU
JF lO/26/92 3/23/93 6/1/93 ll/6/95 MM lO/9/92 3/15/93 6/ll/93 2/18/94 ES 7/2/92 l/5/93 2/5/93 3/24/94 RS 11/13/92 4/19/93 7/23/93 lO/3/g5 Survival Survival LDH Na Stage Gender ~rom D from BEC2 MB 38.63 27.63 153 135 LD
JE 44 90 34.53 164 141 LD M
JF 36.87 29.60 226 126 ED F
MM 16.57 8.40 368 139 ED F
ES 21.00 13.73 196 139 ED F
RS 35.13 26.73 165 138 LD M
BEC2~BCG
25~ The vaccine i8 injected intradermally into multiple rotated sites on weeks O, 2, 4, 6, and 10.
The initial immunization contained 2.5 mg BEC2 mixed with 1 x 107 CFU o~ BCG. In the qetting of grade III local CA 02243~09 l998-07-l~
toxicity, subsequent doses oE BCG are attenuated 3-~old (i.e. 3 x 106 CFU, 1 x 106 CFU, 3 x 105 CFIJ, 1 x 105 CFU, 3 x 104 CFU, 1 x 104 CFU). Patients with a positive PPD (or a history of a positive PPD), are started at an attenuated BCG
dose of 3 x 106 CFU.
Experimental reQult~:
Disease recurrence and overall survival o~ SCLC ~atients All SCLC patients immunized with BEC2/BCG developed anti-BEC2 and 1/6 developed anti-GD3 antibodies. The median survival was not reached but will exceed 36 months.
15 For survival comparison, 34 patients who had major tumor responses ai~ter at least 6 cycles of chemotherapy -/+
radiation served as reference group. There were no differences in prognostic characteristics between the rei~erence group and patients given BEC2/BCG. Median survival 20 of the re~erence group and patients given BEC2/BCG is 16.2 months (95~ Cl; 14-21 months). Using a log-rank test, a signlficant di~ erence in Kaplan-Meier survival curves was demonstrated (p=O.019). At 36 months the survival rate was 18~ (95~6 Cl; 8-35~) for the ref~erence group versus 67~ (95 25 Cli 26-92~) ~or patients receiving BEC2/BCG (Figure 4).
Patients immunized with BEC2/BCG showed a long median and excellent 36 months survival.
_ CA 02243~09 1998-07-1~
Third Seri13~ o:E Experiment~:
LONG SURVIVAL IN 15 PATIENTS (PTS) WITH SMALL CELL LUNG
CANCER (SCLC) IMMUNIZED WITH BEC2 PLUS BCG AFTER INITIAL
THERAPY
No chemotherapeutic intervention has substantially improved the survival o~ SCLC pts beyond that achieved over 20 years ago. SCLC patients who had a major response to i~itial therapy were evaluated ~or their ability to be immunized against GD3 ganglioside present on SCLC tumors. Patients were injected intradermally on weeks 0, 2, 4, 6, and 10 with 2.5 mg BEC2, an anti-idiotypic mouse monoclonal antibody (Ab) that mimics GD3 and can induce anti-GD3 Abs.BCG (2 x 107 CFU) was added as an adjuvant; the dose wa~ attenuated ~or skin toxicity 3+,in 14/15 patients o~ 15 patients entered, 3 did not complete ; mml~n; zation due to SCLC
progression. All were evaluated ~or response. Median ~ollow up is 16 months (mo)(range 10 -55 mo). Pt characteristics: median age 61; median KPS 80~; 8 men;
stage: extensive (ED) 8, limited (LD) 7; elevated LDH 5, hyponatremia 3. All developed anti-BEC2 Ab and 1 developed anti-GD3 Ab. Median survival is 20.5 mo. For survival comparison, 31 patients with major tumor responses after at 25- least 6 cycles o~ chemotherapy radiation, of 104 patients entered on our most recently completed SCLC trial, served as ~ a re~erence group. There were no signi~icant dif~erences in prognostic characteristics between this group and patients given BEC2+BCG. Median survival of the re~erence group is 30= 17.9 mo. At 16 mo the predicted overall survival (os) o~
the BEC2 pt was 75.8~ (95~CI; 43.9-92.6~) vs. 51.6~ (37.1-71.~ or the re~erence group. Predicted 08 of ED was 56.3~ at 16 mo; o~ LD, 75~ at 20 mo. Using a log-rank test, a signi~icant di~erence in Kaplan-Meler survival curves was demonstrated (p=0.03). Patients immunized with BEC2+BCG
showed long survival and these encouraging survival results warrant a randomized trial.
and Newman, M.J. Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-l vaccine. J.Immunol. 148:1519-1525, 1992.
CA 02243~09 l998-07-l~
W097/34635 PCT~S97/04S90 Second series of experiment~:
Since the development of combination therapy over 2Q years ago, no chemotherapeutic intervention has substantially prolonged the survival of small cell lung cancer (SCLC) patients from a median survival of 7-16 months.
Material and Method~:
Patients selection Patients who have a major response to initial therapy are injected intradermally on weeks 0, 2,4,6 and 10 with 2.5mg of BEC2 and 2x107 CFU of BCG. The dose of BCG is attenuated for skin toxicity superior or equal to 3. Patients characteristics are a median age of 63 years old, a median KPS of 80~, an elevated LDH2 in two out six patients and hyponatremia in two out six patients. (Table 4) Of 8 SCLC patients, 6 patients are evaluable for response with a median follow up of 36 months (range 16-43 months).
(table 4) Vaccine comPonents BEC2 is purified from virus-free lipoprotein-free cell culture supernatants by Celltech, Ltd (Slough, England) using Protein A affinity chromatography and anion-exchange chromatography. The final product is ~95~ pure as determined by SDS-polyacrylamide gel electrophoresis. BEC2 is supplied in sterile phosphate buffered saline at concentration of 2.5 mg/ml in single-dose vials.
BCG (TheraCys~) is purchased from Connaught Laboratories (Swiftwater, PA) in vials containing 3.4 x 108 colony-forming units (CFU)/vial in a freeze-dried form. Immediately before administration, BCG is reconstltuted with 3.4 ml of sterile diluent and then further diluted in normal saline if necessary. Vaccine is prepared by mixing 1 ml of BEC2 (2.5 CA 02243509 l998-07-l5 W097/34635 . PCT~S97/04590 mg) with 0.1 ml of the appropriate dilution of BCG
immediately prior to administration.
Table 4. Immunization of small cell lung cancer patients with BEC2 and BCG
Date o~ Dx Complete Rx Date BEC2 Last FU
JF lO/26/92 3/23/93 6/1/93 ll/6/95 MM lO/9/92 3/15/93 6/ll/93 2/18/94 ES 7/2/92 l/5/93 2/5/93 3/24/94 RS 11/13/92 4/19/93 7/23/93 lO/3/g5 Survival Survival LDH Na Stage Gender ~rom D from BEC2 MB 38.63 27.63 153 135 LD
JE 44 90 34.53 164 141 LD M
JF 36.87 29.60 226 126 ED F
MM 16.57 8.40 368 139 ED F
ES 21.00 13.73 196 139 ED F
RS 35.13 26.73 165 138 LD M
BEC2~BCG
25~ The vaccine i8 injected intradermally into multiple rotated sites on weeks O, 2, 4, 6, and 10.
The initial immunization contained 2.5 mg BEC2 mixed with 1 x 107 CFU o~ BCG. In the qetting of grade III local CA 02243~09 l998-07-l~
toxicity, subsequent doses oE BCG are attenuated 3-~old (i.e. 3 x 106 CFU, 1 x 106 CFU, 3 x 105 CFIJ, 1 x 105 CFU, 3 x 104 CFU, 1 x 104 CFU). Patients with a positive PPD (or a history of a positive PPD), are started at an attenuated BCG
dose of 3 x 106 CFU.
Experimental reQult~:
Disease recurrence and overall survival o~ SCLC ~atients All SCLC patients immunized with BEC2/BCG developed anti-BEC2 and 1/6 developed anti-GD3 antibodies. The median survival was not reached but will exceed 36 months.
15 For survival comparison, 34 patients who had major tumor responses ai~ter at least 6 cycles of chemotherapy -/+
radiation served as reference group. There were no differences in prognostic characteristics between the rei~erence group and patients given BEC2/BCG. Median survival 20 of the re~erence group and patients given BEC2/BCG is 16.2 months (95~ Cl; 14-21 months). Using a log-rank test, a signlficant di~ erence in Kaplan-Meier survival curves was demonstrated (p=O.019). At 36 months the survival rate was 18~ (95~6 Cl; 8-35~) for the ref~erence group versus 67~ (95 25 Cli 26-92~) ~or patients receiving BEC2/BCG (Figure 4).
Patients immunized with BEC2/BCG showed a long median and excellent 36 months survival.
_ CA 02243~09 1998-07-1~
Third Seri13~ o:E Experiment~:
LONG SURVIVAL IN 15 PATIENTS (PTS) WITH SMALL CELL LUNG
CANCER (SCLC) IMMUNIZED WITH BEC2 PLUS BCG AFTER INITIAL
THERAPY
No chemotherapeutic intervention has substantially improved the survival o~ SCLC pts beyond that achieved over 20 years ago. SCLC patients who had a major response to i~itial therapy were evaluated ~or their ability to be immunized against GD3 ganglioside present on SCLC tumors. Patients were injected intradermally on weeks 0, 2, 4, 6, and 10 with 2.5 mg BEC2, an anti-idiotypic mouse monoclonal antibody (Ab) that mimics GD3 and can induce anti-GD3 Abs.BCG (2 x 107 CFU) was added as an adjuvant; the dose wa~ attenuated ~or skin toxicity 3+,in 14/15 patients o~ 15 patients entered, 3 did not complete ; mml~n; zation due to SCLC
progression. All were evaluated ~or response. Median ~ollow up is 16 months (mo)(range 10 -55 mo). Pt characteristics: median age 61; median KPS 80~; 8 men;
stage: extensive (ED) 8, limited (LD) 7; elevated LDH 5, hyponatremia 3. All developed anti-BEC2 Ab and 1 developed anti-GD3 Ab. Median survival is 20.5 mo. For survival comparison, 31 patients with major tumor responses after at 25- least 6 cycles o~ chemotherapy radiation, of 104 patients entered on our most recently completed SCLC trial, served as ~ a re~erence group. There were no signi~icant dif~erences in prognostic characteristics between this group and patients given BEC2+BCG. Median survival of the re~erence group is 30= 17.9 mo. At 16 mo the predicted overall survival (os) o~
the BEC2 pt was 75.8~ (95~CI; 43.9-92.6~) vs. 51.6~ (37.1-71.~ or the re~erence group. Predicted 08 of ED was 56.3~ at 16 mo; o~ LD, 75~ at 20 mo. Using a log-rank test, a signi~icant di~erence in Kaplan-Meler survival curves was demonstrated (p=0.03). Patients immunized with BEC2+BCG
showed long survival and these encouraging survival results warrant a randomized trial.
Claims (21)
1. A vaccine comprising an effective amount of anti-idiotypic monoclonal antibody, designated BEC2, an effective amount of an appropriate adjuvant and a pharmaceutically acceptable vehicle.
2. A vaccine of claim 1, wherein the effective amount of BEC2 ranges from about 0.5 mg to 25 mg.
3. A vaccine of claim 2, wherein the effective amount of BEC 2 is 2.5 mg.
4. A vaccine of claim 1, wherein the appropriate adjuvant is BCG.
5. A vaccine of claim 4, wherein the BCG is administered in attenuated doses.
6. A vaccine of claim 1, wherein the vaccine is administered in at least one site on definite time intervals.
7. A vaccine of claim 6, wherein the vaccine is administered in one or more sites.
8. A vaccine of claim 1, wherein the administration is effected intradermally, subcutaneously, parenterally, or intravenously.
9. A vaccine of claim 8, wherein the administration is effected intradermally.
10. A method for stimulating and enhancing in a subject production of antibodies which recognize GD3 ganglioside comprising administering an effective amount of the vaccine of claim 1.
11. A method for treating melanoma in a subject comprising administering to the subject an effective amount of the vaccine of claim 1.
12. A method for preventing the recurrence of cancer comprising administering to a melanoma subject an effective amount of the vaccine of claim 1.
13. A method for increasing the survival rate of small cell lung cancer patients comprising administering to small cell lung cancer patients an effective amount of the vaccine of claim 1.
14. A method of claim 13, wherein the patients are treated with a chemotherapeutic agent.
15. A method of claim 10, 11, 12, 13 or 14, wherein the effective amount of BEC2 ranges from about 0.5 mg to 25 mg.
16. A method of claim 15, wherein the effective amount of BEC 2 is 2.5 mg.
17. A method of claim 10, 11, 12, 13 or 14, wherein the appropriate adjuvant is BCG.
18. A method of claim 18, wherein the BCG is administered in attenuated.
19. A method of claim 10, 11, 12, 13 or 14, wherein the vaccine is administered in at least one site on definite time intervals.
20. A method of claim 10, 11, 12, 13 or 14, wherein the administration is effected intradermally, subcutaneously, parenterally or intravenously.
21. A vaccine of claim 20, wherein the administration is effected intradermally.
Applications Claiming Priority (2)
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US08/619,217 US5792455A (en) | 1996-03-21 | 1996-03-21 | Anti-idiotypic antibody vaccines |
US08/619,217 | 1996-03-21 |
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CA2243509A1 true CA2243509A1 (en) | 1997-09-25 |
Family
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CA002243509A Abandoned CA2243509A1 (en) | 1996-03-21 | 1997-03-21 | Anti-idiotypic antibody vaccines |
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US (1) | US5792455A (en) |
EP (1) | EP0888126A4 (en) |
JP (1) | JP2000507937A (en) |
AU (1) | AU732551B2 (en) |
CA (1) | CA2243509A1 (en) |
WO (1) | WO1997034635A1 (en) |
Families Citing this family (9)
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US6432402B1 (en) | 1989-05-25 | 2002-08-13 | Sloan-Kettering Institute For Cancer Research | Anti-idiotypic antibody which induces an immune response against a glycosphingolipid and use thereof |
US6805862B1 (en) | 1989-05-25 | 2004-10-19 | Sloan-Kattering Institute For Cancer Research | Anti-idiotypic antibody which induces an immune response against a glycosphingolipid and use thereof |
US20030035797A1 (en) * | 1992-01-27 | 2003-02-20 | Sloan-Kettering Institute For Cancer Research | Anti-idiotypic antibody which induces an immune response against a glycosphingolipid and use thereof |
CU22702A1 (en) * | 1997-10-21 | 2001-07-31 | Centro Inmunologia Molecular | ANTI - IDIOTIPO MONOCLONAL ANTIBODIES, THEIR USE IN THE ACTIVE IMMUNOTHERAPY OF MALIGNUM TUMORS, HYBRIDOMA THAT PRODUCES THEM AND COMPOSITIONS THAT CONTAIN THEM |
US6689566B1 (en) | 1999-01-14 | 2004-02-10 | Scantibodies Laboratory, Inc. | Methods, kits, and antibodies for detecting parathyroid hormone |
US7820393B2 (en) * | 1999-01-14 | 2010-10-26 | Scantibodies Laboratory, Inc. | Methods, kits and antibodies for detecting parathyroid hormone |
AT500647A1 (en) * | 2002-05-21 | 2006-02-15 | Igeneon Krebs Immuntherapie | USE OF A VACCINATE |
US7696322B2 (en) * | 2003-07-28 | 2010-04-13 | Catalent Pharma Solutions, Inc. | Fusion antibodies |
EP2199301A1 (en) | 2008-12-19 | 2010-06-23 | DKFZ Deutsches Krebsforschungszentrum | Immunogenic polypeptides comprising a scaffold polypeptide and a L2 polypeptide or fragment thereof |
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AU603585B2 (en) * | 1986-05-07 | 1990-11-22 | Sloan-Kettering Institute For Cancer Research | Vaccine for stimulating or enhancing production of antibodies directed against GM2 |
DE69032855T2 (en) * | 1989-05-25 | 1999-08-12 | Sloan Kettering Inst Cancer | ANTIIDIOTYPICAL ANTIBODY INDUCING AN IMMUNE RESPONSE TO A GLYCOSPHINGOLIPID AND ITS USE |
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1996
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- 1997-03-21 JP JP9533732A patent/JP2000507937A/en active Pending
- 1997-03-21 EP EP97916900A patent/EP0888126A4/en not_active Withdrawn
- 1997-03-21 WO PCT/US1997/004590 patent/WO1997034635A1/en not_active Application Discontinuation
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US5792455A (en) | 1998-08-11 |
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